Reagents

Standard chemicals and reagents were purchased from SIGMA, Merck and ROTH.

Name Supplier

Agarose, Electrophoreses Grade PEQLAB

Ammonium peroxodisulfate APS Merck

Ampicilline (aminobenzylpenicilline) ROTH

Bacto-Agar Becton

Bromphenol blue ROTH

BSA ROTH

Cell Proliferation Reagent WST-1 Roche

3,3’-Diaminobenzidine tetrahydochloride DAB SIGMA

Diethylpyrocarbonate DEPC SIGMA

DNA ladder (1 kb) Fermentas

Ethidium bromide ROTH

Fluorescent mounting medium DAKO

Harris hematoxylin solution SIGMA

Human FGF-2 Natu Tec

JetPEI Polyplus 2-Mercaptoethanol SIGMA

Milk powder SIGMA

NaI¹²⁵ Amersham Pharmacia

Ni-NTA sepharose QIAGEN

Normal goat serum Vector

Normal horse serum Vector

N,N,N',N',-Tetramethylethylendiamine TEMED SIGMA

peqGOLD TriFast^TM PEQLAB

Prestained protein ladder, 10-180 kDa Fermentas

RotiPhorese® Gel 40 ROTH

Salmon sperm DNA SIGMA

Tryptone Becton

Tween 20 SIGMA

Yeast Extract Becton

Materials and Methods 34

3.1.2 Chemotherapeutic agents and phoshotyrosine kinase inhibitors

3.1.3 Kits and enzymes

3.1.4 Antibodies

Name Supplier Alexa Fluor 488-conjugated goat anti-rabbit Molecular Probes

Anti-β-actin, mouse monoclonal SIGMA

Anti-bcl-2, mouse monoclonal DAKO

Anti-caspase-7, rabbit polyclonal Cell Signaling

Anti-caspase-3, rabbit polyclonal PharMingen International

Anti-ERGIC, mouse monoclonal H.-P. Hauri, Basel

Anti-FGF2, rabbit polyclonal Sigma

Anti-FGF-BP, mouse monoclonal R&D

Anti-GFP, rabbit polyclonal Clontech

Anti-p38, rabbit polyclonal Cell Signaling

Anti-p42/44, rabbit polyclonal Cell Signaling

Anti-SAPK/JNK, rabbit polyclonal Cell Signaling

Name Supplier Paclitaxel Pharmacy, Philipps-University Medical Center, Marburg Doxorubicin Pharmacy, Philipps-University Medical Center, Marburg Cisplatin Pharmacy, Philipps-University Medical Center, Marburg

rViscumin VISCUM, Bergisch-Gladbach

D-69491 Baxter, Frankfurt/Main

D-70166 Baxter, Frankfurt/Main

Name Supplier

QIAprep Mini-prep Kit QIAGEN

LumiGLO HRP Western blot kit Cell Signaling

NUCLEOBOND Midi-prep Kit Macherey-Nagel

10mM dNTP Mix Fermentas

Rediprime™ II random prime labeling system Amersham Pharmacia Restriction enzymes: EcoRI, XhoI, NotI, HindIII, and XbaI Fermentas

Ribonuclease Inhibitor Fermentas

Taq Polymerase Fermentas

T4 DNA Ligase supplied with 10x Ligation Buffer Fermentas Transcriptor Reverse Transcriptase supplied with 10x Reaction Buffer Roche

Materials and Methods 35

Anti-phospho p38, rabbit polyclonal Cell Signaling

Anti-phospho p42/p44, rabbit polyclonal Cell Signaling Anti-phospho SAPK/JNK, rabbit polyclonal Cell Signaling

Biotinylated horse anti-mouse IgG Vector

Horseradish peroxidase-conjugated donkey anti-mouse antibodies Amersham Pharmacia Horseradish peroxidase-conjugated donkey anti-rabbit antibodies Amersham Pharmacia

Texas Red-conjugated horse anti-mouse Vector

3.1.5 Oligonucleotides and primers

All oligonucleotides and primers were purchased from MWG BIOTECH, Germany.

Name Sequence FGF-BP_1_f 5'-GGGAATTCATGAAGATCTGTAGCCTCAC-3' FGF-BP_1_r 5'-CCGCTCGAGGCATGACGTGTCCTGCAC-3'

BP-N215_1_f 5'- GGGAATTCATGAAGATCTGTAGCCTCAC-3'

BP-N215_1_r 5'-CCGCTCGAGTCCACAGAACTCCAGGGC-3' BP-N150_1_f 5'-GGGAATTCATGAAGATCTGTAGCCTCAC-3' BP-N150_1_r 5'-CCGCTCGAGTAGCTTAAGACTGGATTCTG-3' BP-N99_1_f 5'-GGGAATTCATGAAGATCTGTAGCCTCAC-3' BP-N99_1_r 5'-CCGCTCGAGATTGCCAGCAAAGACACA-3' BP-C91_1_f 5'-AAGGAAAAAAGCGGCCGCCGAATTTTCCTGTGTCTTTGC-3' BP-C91_1_r 5'-CCGCTCGAGGCATGACGTGTCCTGCAC-3' BP-C146_1_f 5'-AAGGAAAAAAGCGGCCGCCTCCAGTCTTAAGCTAGTCAG-3' BP-C146_1_r 5'-CCGCTCGAGGCATGACGTGTCCTGCAC-3' FGF-BP_2_f 5'-CCCAAGCTTATGAAGATCTGTAGCCTCAC-3' FGF-BP_2_r 5'-GCTCTAGATTAGCATGACGTGTCCTGCAC-3' BP-N215_2_f 5'-CCCAAGCTTATGAAGATCTGTAGCCTCAC-3' BP-N215_2_r 5'-GCTCTAGTATATCCACAGAACTCCAGGGC-3' BP-N150_2_f 5'-CCCAAGCTTATGAAGATCTGTAGCCTCAC-3' BP-N150_2_r 5'-GCTCTAGATTATAGCTTAAGACTGGATTCTG-3' BP-N99_2_f 5'-CCCAAGCTTATGAAGATCTGTAGCCTCAC-3' BP-N99_2_r 5'-GCTCTAGATTAATTGCCAGCAAAGACACAG-3' BP-C91_2_f 5'-CCCAAGCTTATGAAGATCTGTAGCCTCAC-3' BP-C91_2_r 5'-GCTCTAGATTAGCATGACGTGTCCTGCAC-3' BP-C146_2_f 5'-CCCAAGCTTATGAAGATCTGTAGCCTCAC-3' BP-C146_2_r 5'-GCTCTAGATTAGCATGACGTGTCCTGCAC-3' FGF-2_1_f 5'-CCCAAGCTTGGCGGACAGAAGAGCG-3' FGF-2_1_r 5'-GGAATTCCGAGCTCTTAGCAGACATTGG-3' FGF-2_2_f 5'-CCCAAGCTTGGCGGACAGAAGAGCG-3' FGF-2_2_r 5'-GCTCTAGATCAGCTCTTAGCAGACATTGG-3' RT-FGFr1_f 5'-CAAGGTCCGTTATGCCACCTG-3' RT-FGFr1_r 5'-CATCACTGCCGGCCTCTCTTC-3' FGFr1_South 5'-CTGCGTCCTCAAAGGAGAC-3' BP_South_1 5'-GGAATTCATGAAAGTGGTCTCAGAACAAAA-3'

BP_South_2 5'-GGAATTCATGTCCAGTCTTAAGCTAGTCAG -3'

Materials and Methods 36

3.1.6 Bacterial cells and Vectors

3.1.7 Tissue culture media and reagents

3.1.8 Cell lines

Name Supplier

Escherichia coli DH5α Gibco BRL

pRc/CMV expression plasmid Invitrogen

Derivative pcDNA 3.1-based plasmid containing CFP

W. Chubanov (Department of Pharmacology and Toxicology, Philipps-University, Marburg) Derivative pcDNA 3.1-based plasmid

containing YFP

W. Chubanov (Department of Pharmacology and Toxicology, Philipps-University, Marburg)

Name Supplier Iscove’s Modified Eagle’s Medium (IMDM) supplemented with glutamine PAA SF 900 II supplemented with glutamine Life Technologies

Fetal calf serum (FCS) PAA

Trypsin/EDTA PAA

Phosphate buffered saline (PBS) PAA

Geniticin® (G418 Sulphate) CALBIOCHEM

Cell line Type Source

COS7 Simian fibroblasts cells American type culture collection (ATCC) SW-3 Adrenal carcinoma cell line American type culture collection (ATCC) SKOV-3 Ovarian carcinoma cell line American type culture collection (ATCC) HepG2 Hepatoblastoma cell line American type culture collection (ATCC) SF-9 Insect cells American type culture collection (ATCC)

Materials and Methods 37

3.1.9 Equipment, devices and working materials

3.1.10 Standard solutions, buffers and bacterial growth media

Cell culture freezing medium

DMSO 5% (w/v)

FCS 20% (v/v)

IMDM Ad 100% (v/v)

Name Supplier Adjustable air-displacement pipettes Pipetman® Gilson

Autoclave Varioklav® H+P Labortechnik

Biomax MS films Kodak

Centricon-10 vials Amicon

Centrifuge Biofuge Heraeus

Centrifuge Megafuge 1.0 R Heraeus

Centrifuge Sorvall RC 5B Du Pont

CO2-incubator Hera Cell Heraeus

Confocal laser scanning inverted microscope LSM 510 META Carl Zeiss

Digital Camera Digital Science DC 120 Zoom Kodak

Dounce homogenizer B.Braun

ELISA reader BIO-TEK

Exposition chamber Hypercassette™ Amersham Pharmacia

Hyperfilm ECL Amersham Pharmacia

Incubatior Function Line Heraeus

Laminar flow hood Hera Safe Heraeus

Light microscope Wilovert A Hund

Magnetmixer Variomag® H+P Labortechnik

Microspin column (gel filtration) BIO-RAD

Nitrocellulose membrane Schleicher & Schuell

Nylon membrane (HyBond N) Amersham Pharmacia

PCR machine T3 Thermal cycler Biometra

pH-meter Radiometer

Poly-Prep® Chromatography columns BIO-RAD

Orbital Shaker Forma Scientific

Refrigerator -86C Freezer Forma Scientific

Rotor SS-34 Sorvall

γ-Sintillation counter Packard

Ultracentrifuge L7-55 Beckman

UV-Bank UV Transilluminator 2000 Bio-Rad

Vortex Genie 2 Scientific Industries

XCell II Blot Module NOVEX

Materials and Methods 38

10x PBS (Phosphate-buffered saline)

NaCl 1.5 M

Na2HPO4 52 mM

KH2PO4 17 mM

PBS-T

NaCl 150 mM

Na2HPO4 5.2 mM

KH2PO4 1.7 mM

Tween 20 0.05% (v/v)

10xTBS-T

Tris-HCl 1 M

NaCl 1.5 M

Tween 20 0.5% (v/v)

Denaturating lysis buffer

SDS 4 % (v/v)

2-Mercaptoethanol 40 % (v/v)

Glycerol 20 % (v/v)

Bromphenol blue 0.01 % (w/v)

10x SDS electrophoresis buffer

Tris-HCl 2.9 % (w/v)

Glycine 14.4 % (w/v)

SDS 1 % (w/v)

10x Electrophoresis transfer buffer

Tris-HCl 2.9 % (w/v)

Glycine 14.4 % (w/v)

pH adjusted to 8.5

10x Western blot electrophoresis transfer buffer

Tris-HCl 2.9 % (w/v)

Glycine 14.4 % (w/v)

pH adjusted to 8.5

Stacking gel (4%)

RotiPhorese® Gel 40 (38 % Acrylamid, 2 % Bisacrylamid) 10 % (v/v)

1 M Tris-HCl pH 6,8 12.5 % (v/v)

10% SDS 1 % (v/v)

ddH20 Add 100% (v/v)

Materials and Methods 39

For 4 ml stacking gel (4%):

10 % (w/v) APS 40 µl

TEMED 4 µl

Separating gel (12%)

RotiPhorese® Gel 40 (38 % Acrylamid, 2 % Bisacrylamid) 30 % (v/v)

1 M Tris-HCl pH 8,9 37.5 % (v/v)

10% SDS 1 % (v/v)

ddH20 Add 100% (v/v)

For 12.5 ml separating gel (12%):

10 % (w/v) APS 62.5 µl

TEMED 6.25 µl

Western blot stripping buffer

2-Mercaptoethanol 100 mM

Tris-HCl pH 6.7 2.5 mM

SDS 2% (w/v)

Coomassie blue staining solution

Coomassie blue 0.125% (w/v)

Ethanol 25% (v/v)

Glacial acetic acid 10% (v/v)

Destaining solution

Glacial acetic acid 10% (v/v)

Ethanol 20% (v/v)

Drying solution

Glycerol 7% (v/v)

Methanol 55% (v/v)

Lysis buffer

Guanidinehydrochloride 6 M

Sodium phosphate 0.1 M

Tris-HCl 0.01 M

pH adjusted to 8.0

Elution buffer (Ni-NTA affinity chromatography)

Urea 8 M

Sodium phosphate 0.1 M

Tris-HCl 0.01 M

pH adjusted to 8.0

Materials and Methods 40

10x TAE (Tris/acetate/EDTA) electrophoresis buffer

Tris-HCl 2 M

Glacial acetic acid 1 M

EDTA (pH 8.0) 0.05 M

10 x DNA loading buffer

Glycerol 50% (v/v)

EDTA 0.1 M

SDS 0.1% (w/v)

Bromophenol blue 0.05% (w/v)

Xylene cyanol FF 0.05% (w/v)

Southern blot denaturation solution

NaCl 1.0 M

NaOH 0.5 M

Southern blot neutralization solution

NaCl 1.5 M

Tris-HCl 0.5 M (pH 7.2)

EDTA 1 mM

20× SSC

NaCl 3.0 M

Sodium citrat 0.3 M

50x Denhardt’s reagent

Ficoll 400 1% (w/v)

Polyvinylpyrrolidone 1% (w/v)

Bovine serum albumin (BSA) 1% (w/v)

Southern prehybridization solution

SSC 5x

Denhardt 5x

SDS 0.5%

Add 100µl denaturated salmon sperm (10 mg/ml)/10 ml solution

Hydridization membrane washing solution

SSC 2x

SDS 0.1% (w/v)

Materials and Methods 41

SOB medium

Tryptone 2 % (w/v)

Yeast extract 0.5 % (w/v)

NaCl 10 mM

KCl 2.5 mM

MgCl2 10 mM

MgSO4 10 mM

Medium was prepared and autoclaved without magnesium. Filter sterilized stock solutions of 1 M MgCl2 and 1 M MgSO4 were added directly before use.

Transformation buffer

PIPES 10 mM

Yeast extract 15 mM

CaCl2 10 mM

KCl 250 mM

MnCl2 55 mM

The buffer was made without manganese, pH was adjusted to 6.7 and then MnCl2 was added.

LB Medium

Tryptone 1 % (w/v)

Yeast extract 0.5 % (w/v)

NaCl 1 % (w/v)

pH adjusted to 7.5

LB Plates with ampicilline LB medium

Bacto-Agar 1.5 % (w/v)

Ampicilline 100 µg/ml

Im Dokument The Fibroblast Growth Factor-binding Protein (FGF-BP) and the Human Epidermal Growth Factor Receptor-2 (HER-2): functional studies on two gene products relevant in Ovarian cancer (Seite 40-49)